Receptor tyrosine kinases (RTKs) represent a family of cell surface receptors. Both of insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) are type II RTKs that play essential roles in controlling glucose metabolism, cellular growth, proliferation, differentiation, and migration. Aberrant IR or IGF1R signaling causes a number of human diseases, including diabetes and cancers, which makes them promising therapeutic targets. Despite the functional importance and strong connections with diseases, the exact ligand induced activation mechanism of IR and IGF1R are still unknown, due to the lack of high resolution structures of full-length receptors/ligands complex. The goal of this project is to reveal how ligands (insulin and IGF1, respectively) binds to these two different types of receptors and how ligand binding leads to the receptor activation. Although both the IR and IGF1R share high sequence identity and structural similarity, they have been shown to exhibit great diversification in the ligand binding properties, which is indicative of diverse activation mechanism. Consistently, our preliminary structural work showed that the fully liganded IR dimer is bound by 4 insulin molecules at two distinct types of sites?the well-known site 1 and our newly discovered site 2; whereas, only one IGF1 molecule binds the asymmetric IGF1R dimer, meaning that the binding of IGF1 to IGF1R involves negative cooperativity. We will combine cryo-electron microscopy (cryo-EM), biophysical, biochemical and cellular based approaches to understand the roles of site 1 and 2 insulin binding for IR activation, explain the origin of the negative cooperativity in the binding of IGF1 to IGF1R, and reveal the function of transmembrane domains (TM) of IR and IGF1R in receptor activation. Aim 1 will be focused on the functional and structural analyses of full-length IR in the active state. These studies will reveal the detailed binding mode between insulin and the novel IR site 2, and identify the critical role of each type of insulin binding. In addition, we will test whether IRs with different number of insulins bound exhibit different levels of activities and trigger distinct downstream signaling. Aim 2 will be focused on biochemical and structural analyses of full-length IGF1R in the active state. We will identify the structural requirements for the negative cooperative binding of IGF1 to IGF1R, and explain the functional importance of the negative cooperative for IGF1R activation. Aim 3 will be focused on the direct visualization of the TM-TM interaction in the active state of IR and IGF1R to understand why the TM dimerization in the active state of IR and IGF1R is important for receptor activation.